Encapsulation of enzymes and other biomacromolecules in sol-gel materials has drawn great interest in recent years because of the various potential applications in, for example, biocatalysis, biosensor, and drug release vehicles. Encapsulated enzymes retain the same functionality but usually have higher thermal, storage and operational stability in comparison with their counterparts in solution. However, the apparent activity of an entrapped enzyme is often hindered by internal diffusion, and sometimes, by reduced accessibility in microporous sol-gel matrixes even if the synthesis is optimized to preserve the labile biomolecules.
Recently, chemically inert inorganic oxide sol-gel materials with negligible swelling effects, tunable porosity and high purity have emerged as a new class of host matrixes that are well suited for immobilization of biomolecules under room temperature conditions (Avnir et al. Chem. Mater. 1994, 1605–1614; Yamanaka et al. Chem. Mater. 1992 4:497–500; and Dave et al. Anal. Chem. 1994 66:1120–1127A). Many glucose biosensors based on the entrapment of glucose oxidase or oxidase/peroxidase in sol-gels have been reported (Narang, et al. Anal. Chem. 1994 66:3139–3144; Shtelzer et al. Biotechnol. Appl. Biochem. 1994 19:293–305; and Coche-Guérente et al. Chem. Mater. 1997 9:1348–1352).
Immobilization of bioactive substances in mesoporous, host materials via encapsulation of bioagents through a nonsurfactant-mediated sol-gel reaction is described in WO 99/36357. Using this method, it has been shown that enzymes, such as alkaline phosphatase and horseradish peroxidase, entrapped in the nonsurfactant-templated mesoporous, sol-gel matrixes have significantly higher catalytic activities than those in the nontemplated microporous hosts. One of the reasons for this high bioactivity is that substrate and product molecules can diffuse in and out of the mesoporous host matrixes more easily than in conventional microporous materials of typical pore diameters of less than 2 μm.
Since 1992, a great number of papers have also been published on surfactant-templated mesoporous or nanoporous materials. Various ionic and nonionic surfactants have been used as the templates in the hydrothermal synthesis of an ordered MCM-41-type of molecular sieve. Mesoporous sol-gel materials, such as the MCM-41 molecular sieves and the like, are among the ideal host matrixes for immobilizing enzymes because of their large pore volumes and controllable pore sizes with narrow distributions appropriate for inclusion compounds. Various synthetic conditions have been used in an attempt to immobilize enzymes in PEO surfactant-templated sol-gel materials, including using different stoichiometric ratios of water to surfactant to silica precursor in the feed (Attard et al. Nature 1995 378:366; Bagshaw et al. Science 1995 269:124215–17; and Zhao et al. J. Am. Chem. Soc. 1998 120:6024). However, due to the harsh conditions used in the hydrothermal synthesis of highly ordered surfactant-templated matrixes such as MCM-41-type molecular sieves most biomacromolecules cannot survive. Previous studies show that near neutral pH and room temperature conditions are generally required for successful sol-gel immobilization of enzymes. Thus, to date, direct immobilization of active biomacromolecules in such materials has been unsuccessful (Diaz et al. Mol. Catal. B: Enzym. 1996 2:115–126).